The hematopoietic stem cell (HSC) is a pluripotent progenitor cell that has been characterized as a cell that is transplantable, can self-replicate and has multilineage potential. Differentiation of HSCs results in a loss of such multilineage potential, and corresponding lineage commitment. It has been demonstrated that self-renewal of HSC occurs in vivo, as indicated by transplantation studies wherein a single HSC repopulated the marrow of an immunodeficient mouse (Smith, et al., 1991; Osawa, et al., 1996). It has also been demonstrated that hematopoietic stem cells can be infected with recombinant retroviruses, and can serve as cellular targets for gene therapy (Keller and Snodgrass, 1990). (See also, Schmidt-Wolf, I. G., et al., 1991.)
Patients suffering from various cell-based diseases including, but not limited to, myeloproliferative diseases, blood cell proliferative diseases and autoimmune diseases often have an imbalance in the number of cells of particular lineages. In addition, patients undergoing chemotherapy or irradiation often have defective hematopoiesis.
It follows that the modulation of hematopoietic cell processes in patients suffering from any of the above pathological conditions has numerous clinical utilities and that such cells are targets for genetic engineering-based therapies (Wilson, J. D., et al, 1991).
Inhibition of the expression of genes associated with cellular development has been used to modify developmental processes toward directions which are not dependent on the expression of the inhibited gene or genes. Inhibition of genes associated with cellular development has been achieved using antisense technology.
It has been demonstrated that antisense oligonucleotides can be designed to specifically interfere with synthesis of a target protein of interest (Moffat, 1991). Antisense oligonucleotides of 15-20 bases are usually long enough to have one complementary sequence in the mammalian genome. In addition, they hybridize well with their target mRNA (Cohen, et al., 1991).
Due to their hydrophobicity, antisense oligonucleotides interact well with phospholipid membranes (Akhtar, S., et al., 1991), and it has been suggested that following the interaction with the cellular plasma membrane, oligonucleotides are actively transported into living cells (Loke, S. L., et al., 1989; Yakubov, L. A., et al., 1989; Anderson, C. M., et al., 1999).
Inhibition of genes associated with cellular development has been achieved using antisense technology, however, naturally occurring oligonucleotides have a nuclease-sensitive phosphodiester backbone.
Such naturally occurring oligonucleotides may be modified to render them resistant to degradation by nucleases, e.g., by utilizing a methylphosphonate, phosphorothioate or phosphoamidate linkage instead of a phosphodiester one (Spitzer and Eckstein, 1988; Baker, et al, 1990; Hudziak, 1996).
Nonionic methyl-phosphonate analogs were predicted to exhibit increased cellular uptake (Blake, et al., 1985), however, antisense methylphosphonate oligomers were shown to be incapable of inhibiting N-ras expression in vitro (Tidd, et al., 1988), whereas the in vitro translation of several oncogene mRNAs was successfully blocked by phosphodiester and/or phosphorothioate antisense oligonucleotides. See, for example, McManaway, et al., 1990, and Watson, et al., 1991 (c-myc inhibition); Reed, et al., 1990 (bcl-2 inhibition); Calabrett, et al., 1991 (myb inhibition); Szczylik, et al., 1991 (bcr-abl inhibition).
Morpholino oligonucleolides have been demonstrated to exhibit high binding affinity for RNA targets, and the uncharged backbone favors uptake into cells and reduces non-specific binding interactions. (See, e.g., Summerton, et al., 1997).
For therapeutic purposes, it would be desirable to provide a means to modulate hematopoietic stem cell differentiation using an agent which acts specifically on hematopoietic stem cells.